Calmodulin (CaM) acts as the primary cytosolic [Ca2+] sensor for Ca2+ signaling processes as diverse as secretion, motility, transcription, and proliferation. The binding of Ca2+ to EF-hand domains within CaM initiates a conformational change that promotes the interaction of CaM with a number of target proteins, including ion channels, gated pores that conduct ions across cell membranes. CaM modulates the activity of both Ca2+-permeable channels and channels that control the electrochemical driving force for Ca2+ entry, providing a critical feedback mechanism for CaM activity through changes in intracellular [Ca2+]. The Ca2+-dependent opening of small conductance, Ca2+-activated potassium (SK) channels is conferred by the constitutive association of CaM with a binding domain (CaMBD) within each pore- forming subunit of the SK tetramer. As with other ion channels, the structural features of CaM-mediated gating have been gleaned from biochemical and crystallographic studies of CaM bound to a channel fragment including the CaMBD.Acentral question surrounding these studies is whether CaM/CaMBD structures represent functional states in intact channels. To address this issue, we have combined inside-out patch-clamp recordings with optical methods to monitor gating dynamics in functional SK channels. We have site-specifically attached small organic dyes to CaM, and we have incorporated these labeled CaMs into SK channels by taking advantage of a recent finding that SK channels cannot form stable interactions with co-expressed CaMs bearing mutations in the N-terminal EF-hands, allowing the rescue of channel activity in excised patches with exogenously applied CaM. In Specific Aim 1, we will use Fvrster resonance energy transfer (FRET) between fluorescent CaMs co-assembled within the same SK tetramer to determine the structural configuration of CaMs in open and closed SK channels. Because energy transfer between CaMs labeled with donor fluorophores (D-CaMs) and CaMs labeled with acceptor fluorophores (A-CaMs) depends on the distance and orientation between D-CaMs and A-CaMs, FRET reports the spatial arrangement of CaMs within the channel complex. In Specific Aim 2, we will determine the location and state-dependence of CaM/SK contacts by monitoring the quenching of bimane attached to CaM by proximal tryptophans introduced into SK channels. Because the site-specific labeling of CaM and SK residues is based upon closely apposed residues in the CaM/CaMBD x-ray structure, quenching between these residues provides a direct comparison of the CaM/CaMBD and SK channel structures. The techniques developed here provide a general methodology for probing the functional interactions between CaM and its ion channel targets. PUBLIC HEALTH RELEVANCE: Small conductance calcium-activated potassium (SK) channels are regarded as attractive therapeutic targets because of their essential roles in neuronal, smooth muscle, and cardiac physiology. Detailed functional and structural studies of SK channels are critical for the development of potent and selective modulators of SK function. The experiments described within this proposal will interrogate SK structure in functional channels using an approach that can be extended to structural studies in other ion channels.